It has already been reported that various microorganisms produce poly-3-hydroxybutyric acid (PHB) or other poly-3-hydroxyalkanoate (PHA) and accumulate such products therein. Such PHA produced by the microorganisms can be utilized for producing various products. Also the PHA produced by microorganisms, being biodegradable, has the advantage that it can be completely decomposed by the microorganisms. Therefore the PHA produced by microorganisms, when discarded, unlike the various conventional synthesized polymers, would not cause pollution resulting from remaining in the natural environment. Also the PHA produced by microorganisms shows satisfactory affinity to the living tissues and is expected in the applications as the soft material for medical use.
However, for wider application of microorganism-produced PHA, for example for application as functional polymer, PHA having a substituent other than alkyl group in the side chain, namely “unusual PHA”, is anticipated to be extremely useful. Examples of hopeful substituents for this purpose include a group containing an aromatic ring (phenyl group, phenoxy group etc.), an unsaturated hydrocarbon group, an ester group, an allyl group, a cyano group, a halogenated hydrocarbon group and an epoxide present on the side chain. Among these, PHA having an aromatic ring is actively investigated as follows:                (a) PHA containing a phenyl group or a partially substituted group thereof:        
Makromol. Chem. 191, 1957-1965(1990) and Macromolecules, 24, 5256-5260(1991) report that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-phenylvaleric acid as a unit, from 5-phenylvaleric acid as a substrate.
Macromolecules, 29, 1762-1766(1996) reports that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(p-tolyl)valeric acid as a unit, from 5-(p-tolyl)valeric acid as a substrate.
Macromolecules, 32, 2889-2895(1999) reports that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and 3-hydroxy-5-(p-nitrophenyl)valeric acid as units, from 5-(2,4-dinitrophenyl)valeric acid as a substrate.                (b) PHA containing phenoxy group or a partially substituted group thereof:        
Macromol. Chem. Phys., 195, 1665-1672(1994) reports that Pseudomonas oleovorans produces a PHA copolymer containing 3-hydroxy-5-hydroxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid as the units, from 11-phenoxyundecanoic acid as a substrate.
Also Japanese Patent No. 2989175 discloses inventions relating to a homopolymer constituted of a 3-hydroxy-5-(monofluorophenoxy) pentanoate (3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy) pentanoate (3H5(DFP)P) unit, a copolymer containing either a 3H5(MFP)P unit or a 3H5(DFP)P unit or both, a novel strain of Pseudomonas putida capable of producing these polymers, and a method for producing the aforementioned polymers utilizing bacteria of genus Pseudomonas. This patent specification teaches, as the effects of such inventions, that PHA polymer having a phenoxy group substituted with 1 or 2 fluorine atoms at the end of the side chain can be biosynthesized from a long-chain fatty acid having a fluorine substituent and that thus produced PHA has a high melting point and is capable of providing stereoregularity and water repellency while maintaining satisfactory working properties.
In addition to the fluorine-substituted PHA having a fluorine substitution on the aromatic ring in the unit, there are also investigated PHA having a cyano group or a nitro group on the aromatic ring in the unit.
Can. J. Microbiol., 41, 32-43(1995) and Polymer International, 39, 205-213(1996) report production of PHA, containing 3-hydroxy-6-(p-cyanophenoxy) hexanoic acid or 3-hydroxy-6-(p-nitrophenoxy) hexanoic acid as the monomer unit, by Pseudomonas oleovorans ATCC 29347 strain and Pseudomonas putida KT2442 strain, from octanoic acid and 6-(p-cyanophenoxy) hexanoic acid or 6-(p-nitrophenoxy) hexanoic acid as a substrate.
These references relate to PHA having an aromatic ring on the side chain, instead of alkyl groups of the usual PHA, which are effective in obtaining polymer with physical properties resulting from such aromatic ring.
Also as a new category not limited to changes in the physical properties, investigations are also made for producing PHA having an appropriate functional group on the side chain, thereby obtaining PHA with new functions utilizing such substituent.
As a specific method for such purpose, investigations are also made for producing PHA having, in a unit thereof, reactive group such as a bromo group or a vinyl group with a high activity for example in an addition reaction to introduce an arbitrary function group in a side chain of the polymer by a chemical conversion utilizing such active group, in order to obtain PHA of multiple functions.
Macromol. Rapid Commun., 20, 91-94(1999) reports production of PHA having a bromo group in a side chain by Pseudomonas oleovorans, and modifying the side chain with a thiolated product of acetylated maltose thereby synthesizing PHA different in solubility and hydrophilicity.
Polymer, 41, 1703-1709(2000) reports producing PHA, having 3-hydroxyalkenic acid with an unsaturated bond (vinyl group) at an end of a side chain as a monomer unit, by Pseudomonas oleovorans with 10-undecenoic acid as a substrate, followed by an oxidation reaction with potassium permanganate to synthesize 3-hydroxyalkanoic acid having a diol at the end of the side chain, which PHA is reported to show such a change in solubility in solvents, as becoming soluble in polar solvents such as methanol, an acetone-water (80/20, v/v) or dimethylsulfoxide and insoluble in non-polar solvents such as chloroform, tetrahydrofuran or acetone.
Also Macromolecules, 31, 1480-1486(1996) reports production of a polyester, including a unit having vinyl group in a side chain by Pseudomonas oleovorans and epoxylating the vinyl group to obtain a polyester having an epoxy group in the side chain.
Also Polymer, 35, 2090-2097(1994) reports a crosslinking reaction within the polyester molecule utilizing the vinyl group in the side chain of polyester, thereby improving physical properties of polyester.
Macromolecular chemistry, 4, 289-293(2001) reports producing PHA, including 3-hydroxy-10-undecenoic acid as a monomer unit, from 10-undecenoic acid as a substrate, and then executing an oxidation reaction with potassium permanganate to obtain PHA including 3-hydroxy-10-carboxydecanoic acid as a monomer unit, and reports an improvement in a decomposition thereof.
Furthermore, in order to modify physical properties of PHA having an active group in a unit and to actually utilize it as a polymer, it has been studied biosynthesis of a PHA copolymer including a unit having the active group and other units; Macromolecules, 25, 1852-1857(1992) reports production of a PHA copolymer including a 3-hydroxy-ω-bromoalkanoic acid unit and a linear alkanoic acid unit by Pseudomonas oleovorans in the presence of an ω-bromoalkanoic acid such as 11-bromoundecanoic acid, 8-bromooctanoic acid or 6-bromohexanoic acid and n-nonanoic acid.
Such PHA having a highly reactive active group such as a bromo group or a vinyl group can be subjected to introduction of various functional groups or chemical modification, and such a group can be a crosslinking point for a polymer, so that it is very useful means for realizing multiple functions in PHA.
Also technologies related to the present invention include a technology of oxidizing a carbon-carbon double bond with an oxidant to obtain a carboxylic acid (Japanese Patent Application Laid-Open No. S59-190945, J. Chem. Soc., Perkin. Trans. 1, 806(1973), Org. Synth., 4, 698(1963), J. Org. Chem., 46, 19(1981), and J. Am. Chem. Soc., 81, 4273(1959).
On the other hand, active investigations are being made for obtaining a multi-functional PHA from PHA including an ester group in a unit.
Macromol. Chem. Phys., 195, 1405-1421(1994) reports production of a polyhydroxy alkanoate including a unit having an ester group in a side chain, employing Pseudomonas oleovorans as a production microorganism and an alkanoate ester.
Also University of Massachusetts Ph. D. Dissertation Order Number 9132875 (1991) reports production of a polyhydroxy alkanoate including a unit having a benzylester structure, also employing Pseudomonas oleovorans as a production microorganism.
However, the copolymers in the foregoing reports are comprised of a monomer unit having a carboxyl group or an ester group at the end of a side chain and a monomer unit having a linear alkyl group (usual PHA) having a low glass transition temperature. On the other hand, there is no report on copolymers including unusual PHA having on the side chain thereof a substituent other than a linear alkyl group, such as a phenyl structure, a thienyl structure or a cyclohexyl structure. Thus. such polyhydroxy alkanoate and a producing method therefor have been required.
Also PHA having a vinyl group as an active group is a PHA copolymer with a monomer unit having a linear alkyl group(usual PHA), its low glass transition temperature and low melting point are undesirable properties in the working and the use of the polymer.
Because of the above-described situation, there have been a demand for PHA having an active group and a production method therefor, such that PHA can be produced by a microorganism at a high yield, the unit ratio of the active group can be controlled, and its physical properties can be freely regulated not to limit its application as a polymer.